Knockdown of hepatocyte aquaporin-8 by RNA interference induces defective bile canalicular water transport.
Aquaporin-8 (AQP8) water channels, which are expressed in rat hepatocyte bile canalicular membranes, are involved in water transport during bile formation. Nevertheless, there is no conclusive evidence that AQP8 mediates water secretion into the bile canaliculus. In this study, we directly evaluated whether AQP8 gene silencing by RNA interference inhibits canalicular water secretion in the human hepatocyte-derived cell line, HepG2. By RT-PCR and immunoblotting we found that HepG2 cells express AQP8 and by confocal immunofluorescence microscopy that it is localized intracellularly and on the canalicular membrane, as described in rat hepatocytes. We also verified the expression of AQP8 in normal human liver. Forty-eight hours after transfection of HepG2 cells with RNA duplexes targeting two different regions of human AQP8 molecule, the levels of AQP8 protein specifically decreased by 60-70%. We found that AQP8 knockdown cells showed a significant decline in the canalicular volume of approximately 70% (P < 0.01), suggesting an impairment in the basal (nonstimulated) canalicular water movement. We also found that the decreased AQP8 expression inhibited the canalicular water transport in response either to an inward osmotic gradient (-65%, P < 0.05) or to the bile secretory agonist dibutyryl cAMP (-80%, P < 0.05). Our data suggest that AQP8 plays a major role in water transport across canalicular membrane of HepG2 cells and support the notion that defective expression of AQP8 causes bile secretory dysfunction in human hepatocytes.
Larocca
M Cecilia
MC
Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 570, 2000 Rosario, Santa Fe, Argentina.
Soria
Leandro R
LR
Espelt
M Victoria
MV
Lehmann
Guillermo L
GL
Marinelli
Raúl A
RA
D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D001648:Bile Canaliculi / Q000187:drug effects / Q000378:metabolism*; D045744:Cell Line, Tumor; D003563:Cyclic CMP / Q000031:analogs & derivatives / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D009995:Osmosis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D014867:Water / Q000378:metabolism*
D020346:Aquaporins; D034741:RNA, Small Interfering; C108330:aquaporin 8; D014867:Water; D003563:Cyclic CMP; C053441:dibutyryl cyclic-3',5'-cytidine monophosphate